Background: Red-flesh fruit is absent from common sweet orange varieties, but is more preferred by consumers due to its visual attraction and nutritional properties. Our previous researches on a spontaneous red-flesh mutant revealed that the trait is caused by lycopene accumulation and is regulated by both transcriptional and post-transcriptional mechanisms. However, the knowledge on post-transcriptional regulation of lycopene accumulation in fruits is rather limited so far.

Results: We used Illumina sequencing method to identify and quantitatively profile small RNAs on the red-flesh sweet orange mutant and its wild type. We identified 85 known miRNAs belonging to 48 families from sweet orange. Comparative profiling revealed that 51 known miRNAs exhibited significant expression differences between mutant (MT) and wild type (WT). We also identified 12 novel miRNAs by the presence of mature miRNAs and corresponding miRNA*s in the sRNA libraries. Comparative analysis showed that 9 novel miRNAs are differentially expressed between WT and MT. Target predictions of the 60 differential miRNAs resulted 418 target genes in sweet orange. GO and KEGG annotation revealed that high ranked miRNA-target genes are those implicated in transcription regulation, protein modification and photosynthesis. The expression profiles of target genes involved in carotenogenesis and photosynthesis were further confirmed to be complementary to the profiles of corresponding miRNAs in WT and MT.

Conclusion: This study comparatively characterized the miRNAomes between the red-flesh mutant and the wild type, the results lay a foundation for unraveling the miRNA-mediated molecular processes that regulate lycopene accumulation in the sweet orange red-flesh mutant.

Mentions:
Our previous studies showed that the stage of 170 days after flowering (DAF) is the critical stage for the phenotype of color break and global gene expression changes during fruit development in the mutant (Figure 1) [11,13]. In this study, sRNA libraries were generated from fruit pulps of 'Anliu' sweet orange (wild type, WT) and its red-flesh mutant (MT) 'Hong Anliu' at 170 DAF stage. Deep sequencing of small RNA libraries yielded 4,987,484 and 7,163,041 unfiltered sequence reads for WT and MT, respectively. The raw sRNA data were submitted to Gene Expression Omnibus (GEO) under accession no. GSE18207. After discarding low quality sequences containing ambiguous nucleotides, sequences shorter than 18 nucleotides, and sequences counts below 3 in both libraries, 1,790,395 (WT) and 3,345,746 (MT) reliable sequences were remained for analysis. These sequences represented 202,965 and 320,443 unique sRNA sequences in WT and MT, respectively. The distribution of unique sRNA length is summarized in Figure 2. The overall distribution pattern was similar between WT and MT, i.e. the majority of sRNAs (approximately 95%) were 21-24 nt in length with 24-nt sRNA as the major peak.

Mentions:
Our previous studies showed that the stage of 170 days after flowering (DAF) is the critical stage for the phenotype of color break and global gene expression changes during fruit development in the mutant (Figure 1) [11,13]. In this study, sRNA libraries were generated from fruit pulps of 'Anliu' sweet orange (wild type, WT) and its red-flesh mutant (MT) 'Hong Anliu' at 170 DAF stage. Deep sequencing of small RNA libraries yielded 4,987,484 and 7,163,041 unfiltered sequence reads for WT and MT, respectively. The raw sRNA data were submitted to Gene Expression Omnibus (GEO) under accession no. GSE18207. After discarding low quality sequences containing ambiguous nucleotides, sequences shorter than 18 nucleotides, and sequences counts below 3 in both libraries, 1,790,395 (WT) and 3,345,746 (MT) reliable sequences were remained for analysis. These sequences represented 202,965 and 320,443 unique sRNA sequences in WT and MT, respectively. The distribution of unique sRNA length is summarized in Figure 2. The overall distribution pattern was similar between WT and MT, i.e. the majority of sRNAs (approximately 95%) were 21-24 nt in length with 24-nt sRNA as the major peak.

Background: Red-flesh fruit is absent from common sweet orange varieties, but is more preferred by consumers due to its visual attraction and nutritional properties. Our previous researches on a spontaneous red-flesh mutant revealed that the trait is caused by lycopene accumulation and is regulated by both transcriptional and post-transcriptional mechanisms. However, the knowledge on post-transcriptional regulation of lycopene accumulation in fruits is rather limited so far.

Results: We used Illumina sequencing method to identify and quantitatively profile small RNAs on the red-flesh sweet orange mutant and its wild type. We identified 85 known miRNAs belonging to 48 families from sweet orange. Comparative profiling revealed that 51 known miRNAs exhibited significant expression differences between mutant (MT) and wild type (WT). We also identified 12 novel miRNAs by the presence of mature miRNAs and corresponding miRNA*s in the sRNA libraries. Comparative analysis showed that 9 novel miRNAs are differentially expressed between WT and MT. Target predictions of the 60 differential miRNAs resulted 418 target genes in sweet orange. GO and KEGG annotation revealed that high ranked miRNA-target genes are those implicated in transcription regulation, protein modification and photosynthesis. The expression profiles of target genes involved in carotenogenesis and photosynthesis were further confirmed to be complementary to the profiles of corresponding miRNAs in WT and MT.

Conclusion: This study comparatively characterized the miRNAomes between the red-flesh mutant and the wild type, the results lay a foundation for unraveling the miRNA-mediated molecular processes that regulate lycopene accumulation in the sweet orange red-flesh mutant.